Comparison of efficiency and time to regeneration of Agrobacterium-mediated transformation methods in Medicago truncatula

Abstract

Background Tissue culture transformation of plants has an element of art to it, with protocols passed on between labs but often not directly compared. As Medicago truncatula has become popular as a model system for legumes, rapid transformation is critical, and many protocols exist, with varying results. Results The M. truncatula ecotypes, R108 and A17, were utilized to compare the effect of a modification to a previously used protocol based on shoot explants on the percentage of transformed plants produced from calli. This percentage was then compared to that of two additional transformation protocols based on root explants in the R108 ecotype. Variations in embryonic tissue sources, media components, time for transformation, and vectors were analyzed. Conclusions While no A17 transgenic plants were obtained, transgenic plantlets from the R108 ecotype were produced in as little as 4 months with a comparison of the two widely studied ecotypes under a single set of conditions. While the protocols tested gave similar results in percentage of transformed plants produced, considerations of labor and time to transgenics that vary between the root explant protocols tested were discovered. These considerations may influence which protocol to choose for introducing a single transgene versus creating lines with multiple mutations utilizing a CRISPR/Cas9 construct. Electronic supplementary material The online version of this article (10.1186/s13007-019-0404-1) contains supplementary material, which is available to authorized users.

An Optimized Transformation System and Functional Test of CYC-Like TCP Gene CpCYC in Chirita pumila (Gesneriaceae)

“…Mimulus has long been recognized as a classic ecological and evolutionary model system [ 35 ]. Lotus japonicus and Medicago are representatives in addressing some specific events of legumes, such as nodules [ 32 , 33 , 34 ]. Populus and rice have been respectively used as models in trees and grasses [ 28 , 41 ].…”

Correction to: Comparison of efficiency and time to regeneration of Agrobacterium-mediated transformation methods in Medicago truncatula

“…Unfortunately, the original version of the article [1] contained an error. It has been brought to the attention of authors that the R108 ecotype was not derived from A17 but is rather a different subspecies derived from an accession collected in Israel, with a different plastid genome, which likely affects the ability to cross to A17.…”

TML1ANDTML2SYNERGISTICALLY REGULATE NODULATION AND AFFECT ARBUSCULAR MYCORRHIZA INMEDICAGO TRUNCATULA

“…Agrobacterium mediated transformation into root segments of 4-7 day old R108 seedlings and taken through callus and tissue culture following the protocol 2A of (Wen et al, 2019) with the following modifications for selection and rooting: the selection of phosphinothricin (ppt) resistant callus used 2mg/L ppt in the media and 2mg/L NAA was used in RCTM6 medium for rooting. All plants originating from a callus were considered one independent line.…”